Regulation of cellular apoptosis using modulators of β-AR receptors

ABSTRACT

The invention concerns the use of β2-adrenergic modulators for regulating cellular apoptosis.

The present invention relates to the use of β2-adrenergic receptor(β2-AR) modulators for the treatment of pathologies associated with aderegulation of apoptosis.

The term “apoptosis” denotes a particular form of cell death whichdepends on a genetically programmed process. Apoptosis is aphysiological mechanism which occurs normally during the development andlife of an organism. It is, inter alia, involved in the morphologicalchanges of organs during ontogenesis, the regulation of the number ofcells in tissues, the removal of certain sub-populations of lymphocytesin the regulation of the immune system, and the removal of abnormalcells.

Certain diseases are associated with a deregulation of apoptosis. Ifthis is abnormally increased, it leads to excessive cell death (forexample in the case of hepatitis, cardiac ischemia, neurodegenerativediseases and AIDS). If, on the other hand, it is insufficient, it allowsthe survival and proliferation of undesirable cells (for example in thecase of carcinogenesis, auto-immune diseases and certain viralinfections).

Cell death brought about by apoptosis results from the activation ofendogenous endonucleases which destroy the cellular DNA; various factorsresulting in this activation have been described. However, themechanisms governing the mode of action of these factors are stillpoorly understood.

Two proteins, TNF-α and the Fas ligand [ITOH et al., Cell, 66, pp.233-240, (1991); OEHM et al., J. Biol. Chem., 267, pp. 10709-10715,(1992)] play a particularly important role in triggering apoptosisprocesses in many cells. These proteins interact with their respectivereceptors, TNFR [or TNF-αr] and Fas [or FasAg, CD95, APO-1], at the cellsurface. It has been found that the expression of these proteins and/orof their membrane receptors is impaired in certain pathologiesassociated with a deregulation of apoptosis; for example, in hepatitis,an increase in the expression of TNF-α and TNFR has been observed[SPENGLER et al., Cytokine, 8(11), pp. 864-872, (1996)] and likewise forthe expression of the Fas receptors [HIRAMATSU et al., Hepatology, 19,pp. 1354-1350, (1994)]; [MOCHIZUKI et al., J. Hepatology, 24, pp. 1-7,(1996)], which is, in contrast, reduced in the case of hepatocarcinomas[HIGAKI et al., Am. J. Pathol., 149, pp. 429-437, (1996)].

Anti-Fas antibodies having an activity similar to that of FasL are usedin experimental models of apoptosis. Injection of these antibodies intomice brings about a fulminant hepatitis due to a massive apoptosis ofthe hepatocytes, leading to the death of the mice within hours of theinjection [OGASAWARA et al., Nature, 364, pp. 806-809, (1993); NAGATA,Prog. Mol. Subcell. Biol., 16, pp. 87-103, (1996)].

Various in vivo inhibitory factors of apoptosis have been demonstrated.It has been observed, for example, that transgenic mice expressing thehuman Bcl-2 protein [LACRONIQUE et al., Nature Medecine, 2, pp. 80-86,(1996); RODRIGUEZ et al., J. Exp. Med., 183, pp. 1031-1036, (1996)], theT antigen of SV40 [ROUQUET et al., Oncogene, 11, pp. 1061-1067, (1995)],and mice to which protease inhibitors of ICE (interleukin 1β convertingenzyme) type had been administered [ROUQUET et al., Curr. Biol., 6, pp.1192-1195, (1996); RODRIGUEZ et al., J. Exp. Med., 184, pp. 2067-2071,(1996)], or an immunomodulator, linomide [REDONDO et al., J. Clin.Invest., 98, pp. 1245-1252, (1996)], were protected against the hepaticapoptosis induced experimentally with anti-Fas antibodies.

Moreover, the team of the Inventors has previously obtained a transgenicmouse, named F28, which expresses functional human β2-adrenergicreceptors (Hβ2-ARs) [ANDRE et al., Eur. J. Biochem., 241, pp. 417-424,(1996)]. In the F28 mouse, the Hβ2-ARs are expressed in the muscle, theheart, the brain, the lungs and, especially, in the liver, in which thelevel of expression is very high. The expression of the β2-ARs in theliver of the F28 mice is closer to that observed in man than to thatobserved in normal mice.

The Inventors have now observed that when anti-Fas antibodies areadministered to transgenic F28 mice, these mice survive thisadministration, and that their liver shows few apoptotic foci, whereasin normal mice, anti-Fas antibodies administered under the sameconditions bring about a massive apoptosis of the hepatocytes, and thedeath of the animals.

The Inventors have thus put forward the hypothesis of a relationshipbetween the increase in the number of β2-ARs in the liver cells of F28mice, and the resistance of these cells to apoptosis, and haveinvestigated whether or not this resistance could effectively result inan increase in the hepatic β2-adrenergic activity.

Several types of β-adrenergic receptor are known, and have been detectedin greater or lesser amounts in various tissues of the body. Theβ1-adrenergic receptors (β1-ARs) and/or β2-adrenergic receptors (β2-ARs)are present in many tissues such as, for example, heart, kidney, lung,liver, brain, adipose tissue, smooth muscle, striated muscle, the cellsof the immune system, etc. The β3-adrenergic receptors (or β3-ARs),which are present in smaller number than the above receptors, have beendetected in the colon, the heart, the kidneys, the lungs, the liver, thebrain, muscle and in particular in adipose tissue. A fourth type of β-ARhas been described recently in the human heart [KAUMANN and MOLENAAR,Naunyn-Schmiedeberg's Arch. Pharmacol., 355(6), pp. 667-681, (1997),KAUMANN and LYNHAM, Br. J. Pharmacol., 120(7), pp. 1187-1189, (1997)].

The β-adrenergic receptors intervene in stimulation of the cAMPsignaling pathway. According to observations carried out in vitro onvarious types of cell, it appears that the activation of adenylatecyclase and/or the stimulation of the cyclic AMP (cAMP) signalingpathway have variable effects on apoptosis; for example, apoptosis isstimulated in the thymocytes [KIZAKI et al., Cytokine, 5, pp. 342-347,(1993); MENTZ et al., Eur. J. Immunol., 25, pp. 1798-1801, (1995)], ingranular cells [AHARONI et al., Exp. Cell. Res., 218, pp. 271-282,(1995)] and in B cells [LOMO et al., J. Immunol., 154, pp. 1634-1643,(1995); BAIXERAS et al., Scand. J. Immunol., 43, pp. 406-412, (1996)],whereas it is inhibited in T-cell hybridomas [LEE et al., J. Immunol.,151, pp. 5208-5217, (1993); HOSHI et al., Int. Immunol., 6, pp.1081-1089, (1994)], human neutrophils [ROSSI et al., Biochem. Biophys.Res. Commun., 217, pp. 892-899, (1995)], MCF-7 mammary carcinoma cells[BOE et al., Br. J. Cancer, 72, pp. 1151-1159, (1995)] and cells derivedfrom the spinal cord [BERRIDGE et al., Exp. Hematol., 21, pp. 269-276,(1993)].

In order to determine the possible role of the hepatic β2-adrenergicactivity in the resistance to apoptosis, the Inventors administered anagonist specific for the β2-ARs to normal mice, and found that theysurvived the injection of anti-Fas antibodies, and were protectedagainst the hepatic apoptosis induced by these antibodies. They alsofound, on the other hand, that the administration to mice of a β2-ARantagonist inhibited the resistance to apoptosis of F28 mice, or that ofnormal mice treated with the β2-AR agonist.

These results thus show that β2-adrenergic regulation plays a major rolein the physiological control of the apoptosis process in vivo.

A subject of the present invention is the use of a β2-adrenergicmodulator to obtain a medicinal product for regulating cell apoptosis.

The β2-adrenergic activity modulators which can be used in accordancewith the present invention comprise β-AR agonists and antagonists whichare active on the β2-ARs; it is possible, for example, to useisoproterenol as an agonist, or propranolol as an antagonist.

Advantageously, modulators that are specific for the β2-AR receptors canbe used. Agonists that are specific for the β2-ARs are, for example,clenbuterol, salbutamol and procaterol; antagonists that are specificfor the β2-ARs are, for example, ICI 118,551 and butoxamine [BILSKI etal., Br. J. Pharmacol., 69, 292P, (1980); DOOLEY and BITTIGER, J.Pharmacol. Meth., 18, pp. 131-136, (1987); STROSBERG, Recombinant CellSurface Receptors: Focal Point for Therapeutic Intervention, ed. M. Y.Browne, 57-76 (1996), R. G. Landes Company].

Depending on the type of tissue in which it is desired to regulate theapoptosis, and depending on the type of regulation which it is desiredto obtain, i.e. a positive regulation leading to an increase inapoptosis, or a negative regulation leading to a decrease in apoptosis,a β2-AR agonist or antagonist will be chosen.

For example:

In the liver, to inhibit cell apoptosis, for example in the context oftreating hepatitis, a β2-AR agonist will be used; in contrast, if it isdesired to stimulate apoptosis, for example to destroy cancer cells, aβ2-AR antagonist will be used.

In the lung, if it is desired, for example, to stimulate the apoptosisof cancer cells in the case of adenocarcinomas, a β2-AR antagonist willbe used.

In the heart, to inhibit cell apoptosis in the context of treatingcardiac ischemias, a β2-AR agonist will be used.

In the brain, it will be possible via the same approach to reduce theexcessive apoptosis observed in neurodegenerative diseases, or, incontrast, to stimulate apoptosis in the case of cancers.

The present invention will be understood more clearly with the aid ofthe rest of the description which follows, which refers to non-limitingexamples illustrating the demonstration of the role of β2-ARs in theprevention of hepatic apoptosis, and the use of β2-AR agonists toinhibit this apoptosis.

EXAMPLE 1 Demonstration of the Resistance of F28 Mice to HepaticApoptosis and of the Protective Effect of Clenbuterol

In a first series of experiments, carried out on groups of 3 to 4animals, 12 transgenic F28 mice (described by ANDRE et al., [Eur. J.Biochem., 241, pp. 417-424 (1996)], and 10 C57B1/6 control mice(supplied by IFFA CREDO Lyon, France) received, via intravenousinjection, 15 μg of an anti-Fas monoclonal antibody (Jo2 antibody;PHARMINGEN) diluted in 200 μl of physiological saline.

In a second series of experiments, also carried out on groups of 3 to 4animals, 12 F28 mice and 10 C57B1/6 mice received a pretreatment withclenbuterol, administered orally (by addition of 1.3 mg/l to thedrinking water) 17 hours before the intravenous injection of theanti-Fas monoclonal antibody.

The percentage of surviving mice in each batch, as a function of time(in hours) after the injection of the anti-Fas monoclonal antibody, isillustrated in FIG. 1.

Key to FIG. 1:

C57B1/6 mice not treated with clenbuterol=

C57B1/6 mice treated with clenbuterol=∘

F28 mice not treated with clenbuterol=□

F28 mice treated with clenbuterol=.

These results show that in the absence of pretreatment with clenbuterol,all of the F28 mice survived the injection of the anti-Fas antibody,whereas most of the control mice were dead within 5 hours of theinjection, and none survived longer than 24 hours.

After treatment with clenbuterol, most of the control mice survive theinjection of the anti-Fas antibody; this treatment does not modify thesurvival of the F28 mice.

In each of the groups, the livers of a number of animals were removed tocarry out a histological study. In the case of the animals not treatedwith clenbuterol, hemorrhagic lesions and a massive apoptosis areobserved in the control mice; in the F28 mice, only a few rare apoptoticfoci are observed. In the case of the animals treated with clenbuterol,far fewer apoptotic foci are observed in the treated control mice thanin the untreated control mice. No apoptotic lesions are visible in thetreated F28 mice.

EXAMPLE 2 Inhibition with Propranolol of the Protection Conferred byClenbuterol—Demonstration of a Dose-Dependent Effect of Clenbuterol

In order to confirm that the protection conferred by clenbuterol didindeed involve the β2-ARs, experiments were carried out in the presenceof the β-adrenergic antagonist, propranolol.

C57BL/6 mice divided into 5 batches of 3 received the followingtreatments:

Batch 1: Intravenous injection of 15 μg of anti-Fas Jo2 monoclonalantibodies, without pretreatment;

Batch 2: Intravenous injection of 15 μg of anti-Fas Jo2 monoclonalantibodies, after 18 hours of treatment with 1.3 mg/l of clenbuterol;

Batch 3: Intravenous injection of 15 μg of anti-Fas Jo2 monoclonalantibodies, after a pretreatment for 48 hours with 7 mg/l of propranololand 17 hours with 7 mg/l of propranolol plus 1.3 mg/l of clenbuterol;

Batch 4: Injection of anti-Fas Jo2 monoclonal antibodies, aftertreatment for 18 hours with 2.6 mg/l of clenbuterol;

Batch 5: Injection of anti-Fas Jo2 monoclonal antibodies, after apretreatment for 48 hours with 7 mg/l of propranolol and 18 hours with 7mg/l of propranolol plus 2.6 mg/l of clenbuterol.

The injection of the anti-Fas monoclonal antibody and the pretreatmentwith clenbuterol are carried out as described for Example 1 above. Thepropranolol is administered, like the clenbuterol, orally by addition tothe drinking water.

The percentage of surviving mice in each batch, as a function of time(in minutes) after the injection of the anti-Fas monoclonal antibody, isillustrated by FIGS. 2a and 2 b.

Key to FIG. 2a:

Batch 1: ▪

Batch 2: 

Batch 3: ∘

Key to FIG. 2b:

Batch 1: ▪

Batch 4: 

Batch 5: ∘

These results show that the protection conferred by clenbuterol can beinhibited by propranolol (totally for a clenbuterol dose of 1.3 mg/l,and partially for a clenbuterol dose of 2.6 mg/l).

In addition, comparison of the results of FIG. 2a with those of FIG. 2bshows that the protection conferred by clenbuterol is dose-dependent.

EXAMPLE 3 Demonstration of a Dose-Dependent Protective Effect ofSalbutamol

C57B1/6 mice received a treatment with salbutamol, administered orally(by addition of 1.4 mg/l or 2.1 mg/l to the drinking water), 17 hoursbefore the intravenous injection of the anti-Fas Jo2 monoclonalantibody, according to the protocol described in Example 1.

The percentage of surviving mice in each batch, as a function of time(in hours) after the injection of the anti-Fas monoclonal antibody, isillustrated by FIG. 3.

Key to FIG. 3:

C57B1/6 mice not treated with salbutamol=Δ

C57B1/6 mice treated with salbutamol (1.4 mg/l)=∘

C57B1/6 mice treated with salbutamol (2.1 mg/l)=▴

These results show that the treatment with salbutamol significantlyretards, in a dose-dependent manner, the appearance of the lethaleffects of the anti-fas antibody.

What is claimed is:
 1. A method of treatment of a patient in need of regulation of cellular apoptosis comprising administration of an effective amount of a β2-AR receptor modulator to a patient in need thereof.
 2. The method of claim 1 wherein the β2-AR receptor modulator is a β-adrenergic agonist.
 3. The method of claim 1 wherein the β2-AR receptor modulator is a β-adrenergic antagonist.
 4. The method of claim 1 wherein the patient is in need of regulation of cellular apoptosis in hepatic cells.
 5. The method of claim 4 wherein the patient is in need of inhibition of cellular apoptosis in hepatic cells.
 6. The method of claim 4 wherein the patient is in need of activation of cellular apoptosis in hepatic cells. 